摘要
目的观察缺氧对内皮细胞早期生长反应因子(Egr-1)表达的影响及晚期糖基化终末产物(AGEs)及其受体(RAGE)对上述效应的可能调控机制。方法以培养小鼠主动脉内皮细胞(MAECs)为模型,根据缺氧时间和缺氧前预处理方式,分为不同缺氧时间组[缺氧0(对照组),5,10,15,30,60,120min组]、anti-AGE预处理组、可溶性RAGE(sRAGE)预处理组和蛋白激酶C(PKC)β抑制剂组,实时定量PCR法检测缺氧后Egr-1mRNA含量、ELISA法检测细胞裂解液中AGEs含量、蛋白印迹法检测细胞膜蛋白中PKC各异构体的活化。结果缺氧15min组Egr-1mRNA表达上调,为对照组的(14.13±0.77)倍(P=0.0000);缺氧10～60min内皮细胞裂解液中AGEs堆积;缺氧15min组MAECs膜蛋白中PKCβⅡ含量为(30.57±1.86),显著高于对照组的含量(9.22±1.05)(P=0.002);anti-AGE、sRAGE和PKCβ抑制剂预处理减少缺氧诱导的Egr-1 mRNA表达上调(P=0.0000);anti-AGE和sRAGE预处理显著减少缺氧对PKCβⅡ的活化(P=0.0000)。结论急性缺氧诱导内皮细胞AGEs堆积、PKCβⅡ活化和Egr-1mRNA高表达,AGEs-RAGE和PKCβII信号参与急性缺氧后Egr-1诱导表达的调控。
Objective To observe the impact of hypoxia on the expression of early growth response-l（Egr-1） in mouse aortic endothelial cells（MAECs）, and to study the underlying signaling mech- anism involving advanced glycation end-products （AGEs） and its receptor （receptor for AGEs, RAGE）. Methods MAECs were subjected to hypoxia treatment with a （6.0±0.5） %oxygen concentration for va- rious periods （0, 5, 10, 15, 30, 60 or 120 min）, or were first pre-incubated with anti-AGEs or soluble RAGE （sRAGE） for 2 h, or LY379196 for 45 min respectively, and then subsequently exposed to hypoxia for 15 min. Egr-1 mRNA levels were detected by real time PCR （RT-PCR）, AGEs in cell lysate were monitored by ELISA, and PKC isoformates in membrane fraction were examined by western blot. MAECs cultured with normoxia were used as controls. Results With 15-minute hypoxic exposure, Egr- 1 mRNA level in MAECs was （14.13±0.77） folds higher compared with normoxic controls （P=0. 000 0）. Within 10-60 minutes hypoxic exposure, AGEs were accumulated in MAECs lysate. With 15-minute hypoxic exposure, PKCI3II in membrane fraction is （30.57 ±1.86） vs （9.22± 1.05） arbitrary units com- pared with normoxic controls （P=0. 002）. Hypoxia-induced Egr-1 mRNA expression was decreased by pre-incubation with anti-AGE, sRAGE or PKCβII inhibitor （P = 0. 000 0）. Hypoxia-induced PKCI3II activation was attenuated by pre-incubation with anti-AGE or sRAGE （P = 0. 000 0）. Conclusions In MAECs, acute hypoxia induced AGEs accumulation, PKCβII activation and Egr-1 mRNA expression. AGEs-RAGE and PKCI3II signaling pathway were involved in hypoxia-induced Egr-1 mRNA expression. These findings highlight these signaling molecules as potential therapeutic targets to protect tissue injury by acute hypoxia.
出处
《福建医科大学学报》
2012年第4期227-230,共4页
Journal of Fujian Medical University
基金
福建省自然科学基金(2009J01144)
福建省卫生厅医学创新课题(2009-CXB-18)
